The abuse of psychostimulants such as cocaine, amphetamine and methamphetamine represents a major health problem. Understanding the biological substrates underlying psychostimulant abuse and dependency will certainly prove important in developing successful treatment and prevention strategies. The blockade of the dopamine reuptake mechanism (dopamine transporter protein) and the consequent augmentation of dopamine effects is thought to underlie the reinforcing properties of psychostimulants. Yet surprisingly, virtually nothing is known about the cellular and molecular biology of the DA transporter. The present proposal will study in detail the physiology and pharmacology of the human and rat DA transporters, which will be expressed in Xenopus oocytes following injection of human and rat substantia nigra messenger RNA. Additionally, cDNA libraries will be constructed from human and rat messenger RNA, and the oocyte expression system will be sued to isolate DA transporter clones on the basis of eliciting functional responses. Analysis of the clones will provide information about the predicted size and secondary structure of the DA transporters and possible homologies with size and secondary structure of the DA transporters and possible homologies with other proteins. Antisera will be raised against deducted peptide sequences from the DA transporter and used to identify and characterize with respect to molecular weight the DA transporter protein on Western blots. The regional distribution and cellular localization of human and rat DA transporter mRNAs and proteins will be accomplished using Northern blot, Western blot and immunohistochemical analyses. The effects of chronic treatment with cocaine and other dopamine reuptake inhibitors on DA transport gene expression and functional activity will be examined in rats in vivo and in primary neuronal cultures in an effort to address possible mechanisms linked to the clinical development of tolerance after chronic cocaine use. These cellular and molecular biological analyses of the cocaine-binding DA transporter should prove invaluable in our understanding of the biological basis of cocaine abuse.